Echinicola Pacifica Gen. Nov., Sp. Nov., a Novel Flexibacterium Isolated from the Sea Urchin Strongylocentrotus Intermedius

International Journal of Systematic and Evolutionary Microbiology (2006), 56, 953–958 DOI 10.1099/ijs.0.64156-0

Echinicola paciﬁca gen. nov., sp. nov., a novel ﬂexibacterium isolated from the sea urchin Strongylocentrotus intermedius

Olga I. Nedashkovskaya,1 Seung Bum Kim,2 Marc Vancanneyt,3 Anatoly M. Lysenko,4 Dong Sung Shin,2 Myung Soo Park,5 Kang Hyun Lee,6 Won Jin Jung,6 Natalia I. Kalinovskaya,1 Valery V. Mikhailov,1 Kyung Sook Bae6 and Jean Swings3,7

Correspondence 1Paciﬁc Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy Olga I. Nedashkovskaya of Sciences, Pr. 100 Let Vladivostoku 159, 690022, Vladivostok, Russia [email protected] 2,5Department of Microbiology2 and Department of Applied Microbiology, College of Agriculture or and Life Sciences5, Chungnam National University, 220 Gung-dong, Yusong, [email protected] Daejon 305-764, Republic of Korea 3,7BCCM/LMG Bacteria Collection3 and Laboratory of Microbiology7, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium 4Institute of Microbiology of the Russian Academy of Sciences, Pr. 60 let October 7/2, Moscow, 117811, Russia 6Korea Institute of Bioscience and Biotechnology, 52 Oun-dong, Yusong, Daejon 305-333, Republic of Korea

The taxonomic position of three novel marine, heterotrophic, pigmented and agarolytic bacteria with gliding motility, isolated from the sea urchin Strongylocentrotus intermedius, was investigated. 16S rRNA gene sequence analysis revealed that strains KMM 6166, KMM 6172T and KMM 6173 are members of the phylum Bacteroidetes; their nearest neighbours were Belliella baltica and Hongiella marincola (similarities of 94?5 and 93?6 %, respectively). The DNA G+C content of

the strains was 44–45 mol%. The predominant fatty acids were C15 : 0 iso, C16 : 1v5c,C17 : 1 iso

v9c,C17 : 0 iso 3-OH and summed feature 3 (C16 : 1v7c and/or C15 : 0 iso 2-OH). The major respiratory quinone was MK-7. Results of molecular experiments supported by phenotypic and chemotaxonomic data enabled the isolates to be classified as representatives of a novel species in a new genus, for which the name Echinicola pacifica gen. nov., sp. nov. is proposed. Echinicola pacifica is the type species of the genus Echinicola, and its type strain is KMM 6172T (=KCTC 12368T=LMG 23350T).

A phylogenetic survey on the culturable bacteria associated the basis of their significant molecular divergence from with the sea urchin Strongylocentrotus intermedius revealed described taxa. Results of genotypic, chemotaxonomic and rich species diversity. Previously undescribed bacteria phenotypic analyses confirmed that the strains had a distinct belonging to the Proteobacteria, low- and high-G+C- taxonomic position in a new genus. content Gram-positive bacteria and Bacteroidetes were T found. Up to now, only some of the novel species have Strains KMM 6166, KMM 6172 and KMM 6173 were been assigned validly published names (Nedashkovskaya isolated from the sea urchin Strongylocentrotus intermedius et al., 2005a, b, c, d). collected in Troitsa Bay, Gulf of Peter the Great, East Sea (also known as the Sea of Japan), during September 2002. In the present paper, the heterotrophic, Gram-negative, After primary isolation and purification on marine agar pink-coloured, gliding agarolytic strains KMM 6166, KMM 2216 (Difco), the strains were cultivated on the same T 6172 and KMM 6173 were selected for further study on medium at 25 uC for 48 h and stored at 280 uC in marine broth (Difco) supplemented with 20 % (v/v) glycerol. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Echinicola pacifica KMM 6172T, KMM 6166 and Genomic DNA extraction, PCR and 16S rRNA gene KMM 6173 are DQ185611, DQ186987 and DQ186988, respectively. sequencing were carried out as described previously (Kim

Downloaded from www.microbiologyresearch.org by 64156 G 2006 IUMS Printed in Great Britain 953 IP: 193.191.134.1 On: Fri, 21 Oct 2016 07:15:24 O. I. Nedashkovskaya and others et al., 1998). Sequence data obtained were aligned with those Identification System (Microbial ID). The predominant of representative members of the phylum Bacteroidetes cellular fatty acids of strains KMM 6166 and KMM 6172T using PHYDIT version 3.2 (http://plaza.snu.ac.kr/~jchun/ were C15 : 0 iso (17?3–18?0 %), C16 : 1v5c (6?7–7?8 %), C17 : 1 phydit/). Phylogenetic trees were inferred using suitable iso v9c (6?3–6?9 %), C17 : 1v6c (4?3–4?8 %), C15 : 0 iso 3-OH programs of the PHYLIP package (Felsenstein, 1993). (3?4–5?0 %), C17 : 0 iso 3-OH (9?4–10?0 %) and summed Phylogenetic distances were calculated using the Kimura feature 3 (30?7–30?8 %), comprising C16 : 1v7c and/or C15 : 0 two-parameter model (Kimura, 1980) and trees were iso 2-OH (Table 1). Isoprenoid quinones were extracted constructed on the basis of the neighbour-joining (Saitou from lyophilized cells and analysed as described previously & Nei, 1987), maximum-parsimony (Kluge & Farris, 1969) (Nedashkovskaya et al., 2004b). The main isoprenoid and maximum-likelihood (Felsenstein, 1993) algorithms. quinone of the novel isolates was MK-7. Bootstrap analysis was performed with 1000 resampled The absorption spectrum of pigments extracted using 7 : 2 datasets using the SEQBOOT and CONSENSE programs of the (v/v) acetone/methanol was determined between 300 and PHYLIP package. 700 nm with UV spectrophotometer CECIL, CE 7250, Phylogenetic analysis of the almost-complete 16S rRNA 7000 series. Cells produced pink-coloured carotenoid pig- gene sequences revealed that the three sea-urchin isolates ments with maximum absorption at 472?8 nm. occupied a distinct lineage within the phylum Bacteroidetes Physiological and biochemical properties of strains KMM (Fig. 1). The nearest neighbours of the strains studied were T 6166, KMM 6172 and KMM 6173 were examined as des- Belliella baltica BA134T, Hongiella marincola SW-2T and T cribed by Nedashkovskaya et al. (2004a, b). Physiological Cyclobacterium marinum LMG 13164 , with similarity and biochemical properties of KMM 6172T were also deter- values of 94?5, 93?6 and 93?1 %, respectively. mined using the API 20E, API 20NE, API ZYM and API DNA was isolated according to the method of Marmur 50CH galleries (bioMe´rieux) and the Biolog GN2 Micro- (1961) and its G+C content was determined using the plate system according to the manufacturers’ instruc- thermal denaturation method (Marmur & Doty, 1962). The tions. Susceptibility to antibiotics was tested as described G+C content of the DNA of the strains under study was previously (Nedashkovskaya et al., 2004a) using additional discs containing chloramphenicol (30 mg), doxycycline 44?2–44?6 mol%. DNA–DNA hybridization experiments (10 mg) and erythromycin (15 mg). The ability to grow were performed using the method of De Ley et al. (1970). under anaerobic conditions was observed using the Oxoid DNA–DNA hybridization values between strains KMM Anaerobic System. Gliding motility was determined as 6166, KMM 6172T and KMM 6173 were 93–98 %. There- described by Bowman (2000). fore, the strains can be placed in the same species according to criteria defined by Wayne et al. (1987). Strains isolated in this study were Gram-negative, chemo- organotrophic, pink-coloured and motile by gliding. The Analysis of fatty acid methyl esters was carried out accord- main physiological and biochemical characteristics are given ing to the standard protocol of the Sherlock Microbial in Tables 2 and 3 and the species description. The strains tested differed from their nearest neighbour, B. baltica,by their ability to move by gliding, to grow with 12 % NaCl and to produce hydrogen sulfide. Other distinctive features between these taxa included hydrolysis of agar and gelatin, acid production from D-fructose, D-melibiose and L- rhamnose, enzyme activities and utilization of a number of organic compounds (Table 2). The differential features of the strains studied and other related members of the phylum Bacteroidetes are shown in Table 3. It should be noted that the sea-urchin isolates can be clearly distinguished from all close relatives by their moving by means of gliding and their ability to ferment D-glucose. The latter characteristic of the strains, together with the absence of growth under strictly anaerobic conditions, indicates that the strains may grow in a wide range of oxygen concentrations. Phenotypic divergence between the strains studied and their relatives is supported by significant distinctiveness in the cellular fatty Fig. 1. Phylogenetic tree based on the 16S rRNA gene acid profiles (Table 2). For example, the presence of a sequences of KMM strains and representative members of the substantial amount of C15 : 0 3-OH, the absence of C17 : 1 family ‘Flexibacteraceae’. Asterisks indicate branches that were anteiso and very low levels of C15 : 1 iso G fatty acids were T also recovered using maximum-likelihood and maximum-parsimony noted in extracts of KMM 6166 and KMM 6172 , in contrast algorithms. Numbers at nodes indicate bootstrap values (%). Bar, with their nearest neighbour, B. baltica. Consequently, the 0?1 substitutions per nucleotide position. combination of all data obtained allows the strains to be

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Table 1. Cellular fatty acid composition (%) of Echinicola paciﬁca gen. nov., sp. nov. and related taxa of the phylum Bacteroidetes

Taxa: 1, Echinicola paciﬁca gen. nov., sp. nov.; 2, Algoriphagus;3,Aquiﬂexum;4,Belliella;5,Cyclobacterium;6,Hongiella. Data from Brettar et al. (2004a, b); Nedashkovskaya et al. (2004b, 2005e) and this study. Values of less than 1 % for all strains are not shown. Predominant fatty acids are shown in bold. Summed features consist of one or more fatty acids that could not be separated by the Microbial

Identiﬁcation System. Summed feature 3 is C15 : 0 iso 2-OH and/or C16 : 1v7c; summed feature 4 is C17 : 1 iso I and/or C17 : 1 anteiso B.

Fatty acid 1 2 3 4 5 6

C11 : 0 anteiso 2 0?6–2?1 22 2 0?0–0?9

C13 : 1 AT 222<1?4 22

C14 : 0 iso 0?1–0?20?3–1?4 4?8 2?0 2 0?6–2?3

C15 : 1 iso 0?0–0?6 0–2?9 9?410?28?4–8?5 0?2–0?4

C15 : 0 iso 17?3–18?028?4–38?922?620?722?2–25?526?5–32?7

C15 : 0 anteiso 2?1–2?81?6–3?6 18?54?56?3–9?23?7–6?1

C15 : 0 0?8–1?01?0–2?5 2 2?90?0–0?82?7–3?0

C15 : 1v6c 1?1–1?21?0–2?3 2 2?10?5–1?31?1–4?1

C16 : 1v5c 6?7–7?83?5–5?8 2?03?3 2 0?6–1?1

C16 : 1 iso 0?8–1?01?5–3?5 9?5 3?5 2 3?5–6?4

C16 : 0 iso 1?1–1?22?4–7?7 4?2 2?6 2 6?5–12?3

C16 : 0 0?8–0?9 0–2?9 22 0?5–0?7 2

C15 : 0 iso 3-OH 3?4–5?0 1?6–2?91?62?23?5–3?71?8–2?7

C15 : 0 3-OH 2?5–2?6 22222

C17 : 1 anteiso 222?63?7 22

C17 : 1 iso v9c 6?3–6?9 1?5–9?0 5?28?44?3–5?66?0–12?2

C17 : 1 anteiso v9c 221?1 22 0?0–1?0

C17 : 1v8c 2221?2 22

C17 : 1v6c 4?3–4?8 0?5–3?43?0 7?3 1?3–1?4 4?5–7?0

C16 : 0 iso 3-OH 0?6–0?70?9–3?12?01?90?0–1?03?4–4?7

C16 : 0 3-OH 0?9–1?40?5–1?9 2 <1?11?3–1?7 2

C17 : 0 iso 3-OH 9?4–10?05?9–9?2 1?43?2 10?3–10?76?4–6?7

C17 : 0 2-OH 0?40?0–0?6 22 1?5–2?90?4–1?8

C18 : 1v5c 0?2 2221?2–1?4 2

C19 : 1 iso 221?5 22 2 Summed feature 3 30?7–30?819?0–24?66?19?224?3–25?16?0–7?4 Summed feature 4 2 0?9–2?7 22 2?5–4?42?0–2?5 discriminated from their close relatives. Low sequence non-diffusible carotenoid pigments. Chemo-organotrophs. similarities of the strains tested with other members of Positive for cytochrome oxidase, catalase and alkaline the family ‘Flexibacteraceae’ described to date (81?1–92?5%) phosphatase. The major respiratory quinone is MK-7. The demonstrate clearly that the strains isolated in this study main cellular fatty acids are straight-chain unsaturated and represent a novel genus. branched-chain unsaturated fatty acids C15 : 0 iso, C16 : 1v5c, C17 : 1 iso v9c,C17 : 1v6c,C15 : 0 iso 3-OH, C17 : 0 iso 3-OH Thus, the polyphasic data presented in this study support and summed feature 3, comprising C15 : 0 iso 2-OH and/or the conclusion that strains KMM 6166, KMM 6172T and C16 : 1v7c. As determined by 16S rRNA gene sequence KMM 6173 could not be affiliated to any taxa currently analysis, the genus Echinicola is a member of the phylum included in the phylum Bacteroidetes. Based on this fact, it Bacteroidetes. The type species is Echinicola pacifica. is proposed that these strains should be placed in a novel genus as Echinicola pacifica gen. nov., sp. nov. Description of Echinicola pacifica sp. nov. Echinicola pacifica (pa.ci9fi.ca. N.L. fem. adj. pacifica refer- Description of Echinicola gen. nov. ring to the Pacific Ocean, from which the type strain was Echinicola (E.chi.ni.co9la. L. masc. n. echinus -i sea urchin; L. isolated). suff. -cola derived from L. masc. or fem. n. incola a dweller; N.L. fem. n. Echinicola a sea-urchin dweller). Main characteristics are those given for the genus. In addition, cells are 0?3–0?461?2–1?9 mm. On marine Rod-shaped cells, motile by gliding. Gram-negative. Do agar, colonies are circular, 2–3 mm in diameter, convex, not form endospores. Can ferment D-glucose. Produce shiny, smooth, pink-coloured and sunken into agar.

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Table 2. Phenotypic characteristics that differentiate between Echinicola paciﬁca gen. nov., sp. nov. and Belliella baltica BA134T

Taxa: 1, Echinicola paciﬁca (n=3); 2, Belliella baltica BA134T. Data from Brettar et al. (2004a) and this study.

Characteristic 12

Gliding motility + 2 Nitrate reduction 2 +

H2S production + 2 Growth with 12 % NaCl + 2 Esterase (C4), N-acetylglucosaminidase, a-galactosidase, a-mannosidase, a-fucosidase + 2 Hydrolysis of agar + 2 Acid production from: L-Rhamnose, D-fructose + 2 D-Melibiose 2 + Utilization of: Dextrin, a-cyclodextrin, glycogen, N-acetyl-D-glucosamine, D-fructose, L-fucose, a-lactose, + 2 D-mannose, D-melibiose, methyl b-D-glucoside, psicose, D-rafﬁnose, sucrose, turanose, D-galacturonic acid, D-glucuronic acid, alaninamide, L-alanyl glycine, L-aspartic acid, L-rhamnose, L-alanine, L-asparagine, hydroxy-L-proline, L-threonine Acetic acid, a-ketoglutaric acid, a-ketovaleric acid 2 + DNA G+C content (mol%) 44–45 35?4

+ b-Galactosidase-positive. Does not require Na ions or D-cellobiose, D-glucose, D-lactose, D-maltose, D-mannose, sea water for growth. Growth occurs at 6–41 uC. Optimal L-rhamnose, DL-xylose and N-acetylglucosamine. Can oxi- temperature for growth is 25–28 uC. Growth occurs with dize D-galactose and D-sucrose. Does not form acid from 0–12 % NaCl. No flexirubin-type pigments are formed. fucose, melibiose, raffinose, sorbose, glycerol, adonitol, Degrades agar, gelatin (weakly), aesculin, Tween 40 and dulcitol, inositol or mannitol. Can ferment D-glucose. starch. Can decompose Tween 20 and 80. Does not According to the API 20E gallery (bioMe´rieux), the type hydrolyse casein, DNA, cellulose (carboxymethyl-cellulose strain (KMM 6172T) utilizes citrate, forms acid from or filter paper) or chitin. Produces acid from L-arabinose, amygdalin and is negative for arginine dihydrolase, lysine

Table 3. Phenotypic characteristics that differentiate between Echinicola paciﬁca gen. nov., sp. nov. and its close relatives in the family ‘Flexibacteraceae’

Taxa: 1, Echinicola paciﬁca gen. nov., sp. nov.; 2, Algoriphagus;3,Aquiﬂexum;4,Belliella;5,Cyclobacterium;6,Hongiella. Data from Bowman et al. (2003), Brettar et al. (2004a, b), Nedashkovskaya et al. (2004b, 2005e), Van Trappen et al. (2004), Yi & Chun (2004), Yoon et al. (2004, 2005) and this study. Abbreviations: V, variable; ND, Not detected.

Characteristic 1 2 3 4 5 6

Cell morphology Regular rods Regular rods Regular rods Regular rods Ring-like/ Regular rods horseshoe-shaped Cell size (mm) 0?4–0?66 0?3–0?76 0?3–0?66 0?3–0?56 0?3–0?76 0?3–0?66 0?8–2?3 1?0–10?0 1?1–4?8 0?9–3?0 0?8–1?5 0?8–3?0 Gliding motility + 22222 Nitrate reduction 2 V ++ 2 V Fermentation of D-glucose + 22222 Hydrogen sulﬁde + 2 ND ND 22 Salinity range (%) 0–12 0–10 0–6 0–6 0–10 0–8 Growth at 42 uC 2 V 22 V + Hydrolysis of: Agar + V 2222 Gelatin + V + 22 V Starch + V ++ 2 V DNA G+C content (mol%) 44–45 35–42 38?435?4 41–42 38–43

Downloaded from www.microbiologyresearch.org by 956 International Journal of Systematic and Evolutionary Microbiology 56 IP: 193.191.134.1 On: Fri, 21 Oct 2016 07:15:24 Echinicola pacifica gen. nov., sp. nov. decarboxylase and ornithine decarboxylase. Results of References Biolog GN2 (Biolog) testing show that strain KMM 6172T Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov., utilizes a-cyclodextrin, dextrin, glycogen, a-D-glucose, isolated from the surfaces of Antarctic algae, and reclassification of D-fructose, L-fucose, D-galactose, gentibiose, a-lactose, a- Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 D-lactose, lactulose, D-mannose, D-melibiose, methyl b- as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, D-glucoside, psicose, D-raffinose, sucrose, D-trehalose, 1861–1868. turanose, D-galacturonic acid, D-glucuronic acid, a- Bowman, J. P., Nichols, C. M. & Gibson, J. A. E. (2003). 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A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide produces a- and b-galactosidases, alkaline and acid phos- sequences. J Mol Evol 16, 111–120. phatases, esterase (C4), esterase lipase (C8), leucine Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the arylamidase, valine arylamidase, cystine arylamidase, tryp- evolution of anurans. Syst Zool 18, 1–32. sin, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic a- and b-glucosidases, N-acetyl-b-glucosaminidase, a- acid from microorganisms. J Mol Biol 3, 208–218. mannosidase and a-fucosidase, but not lipase (C14) or Marmur, J. & Doty, P. (1962). Determination of the base composition b-glucuronidase. Susceptible to lincomycin. Resistant to of deoxyribonucleic acid from its thermal denaturation temperature. ampicillin, benzylpenicillin, chloramphenicol, doxycycline, J Mol Biol 4, 109–118. erythromycin, gentamicin, kanamycin, carbenicillin, olean- Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Rhee, M. S., Lysenko, domycin, neomycin, polymixin B, streptomycin and A. M., Falsen, E., Frolova, G. M., Mikhailov, V. V. & Bae, K. S. tetracycline. Predominant fatty acids are C15 : 0 iso (2004a). Ulvibacter litoralis gen. nov., sp. nov., a novel member of (17?3–18?0 %), C16 : 1v5c (6?7–7?8 %), C17 : 1 iso v9c the family Flavobacteriaceae isolated from the green alga Ulva (6?3–6?9 %), C17 : 1v6c (4?3–4?8 %), C15 : 0 iso 3-OH fenestrata. Int J Syst Evol Microbiol 54, 119–123. (3?4–5?0 %), C17 : 0 iso 3-OH (9?4–10?0 %) and summed Nedashkovskaya, O. I., Vancanneyt, M., Van Trappen, S. & 7 other authors (2004b). 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Acknowledgements Nedashkovskaya, O. I., Kim, S. B., Lysenko, A. M., Frolova, G. M., Mikhailov, V. V., Bae, K. S., Lee, D. H. & Kim, I. S. This research was supported by grants from the Russian Foundation for (2005b). Gramella echinicola gen. nov., sp. nov., a novel halophi- Basic Research no. 05-04-48211, Presidium of the Far-Eastern Branch lic bacterium of the family Flavobacteriaceae isolated from the of the Russian Academy of Sciences no. 06-04-96067 and Presidium of sea urchin Strongylocentrotus intermedius. Int J Syst Evol Microbiol the Russian Academy of Sciences ‘Molecular and Cell Biology’. 55, 391–394.

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